The ability of ribosomes to maintain the correct translational reading frame is fundamental to the integrity of protein synthesis and to cell growth and viability. However, there are now a number of mostly viral examples in which elongating ribosomes are programmed to shift their translational reading. This process is called programmed ribosomal frameshifting. We have been using the naturally occurring Killer and Ty1 viruses of the yeast Saccharomyces cerevisiae to investigate the cis-acting elements and trans-acting factors that determine the efficiency of programmed ribosomal frameshifting by either one base in the 5'(-1), or one base in the 3'(+1) direction respectively. We have shown that small changes in the ratio of the Gag to Gag-pol proteins synthesized by altering frameshifting efficiencies prevents propagation of these viruses, suggesting that programmed ribosomal frameshifting may present a novel target for antiviral agents against clinically relevant viruses, such as HIV. In addition, this mechanism provides us with a set of novel experimental tools that can be used to probe the molecular mechanisms underlying translational fidelity and the post- transcriptional regulation of gene expression. Our recent investigations into the mechanisms of programmed ribosomal frameshifting suggest that there are three critical factors which can contribute towards determining the efficiency of programmed ribosomal frameshifting. These are 1) the amount of time that a ribosome is paused at a ribosomal frameshift signal; 2) the status of the error sensing mechanism of the ribosome; and 3) the quality of the interactions between tRNAs and ribosomes. In order to refine our understanding of the molecular mechanisms underlying this unique problem in translation, we have identified a unique set of chromosomal mutants called mof (Maintenance Of Frame), discovered drugs that alter the efficiency of -1 ribosomal frameshifting, and developed an in vitro assay which recapitulates the programmed ribosomal frameshifting efficiencies observed in vivo. These tools will be used to probe aspects of ribosome pausing, translational proofreading, and kinetic interactions between tRNAs and the ribosomes in an effort to understand how they contribute to the efficiency of programmed ribosomal frameshifting. The proposed research will allow us to elucidate the various factors that can influence programmed ribosomal frameshifting efficiencies while also serving to classify the mutants and compounds according to their translational defects. This analysis will also serve to develop the proof of principle needed for a rational approach to identify antiviral agents that target ribosomal frameshifting.